Combinatorial control of gene expression is a fundamental mechanism for integrating diverse regulatory pathways within a cell. It is founded on the principles of cooperativity and synergy. Unique arrays of activators bind cooperatively to enhancers and promoters and assemble into nucleoprotein complexes termed enhanceosomes. Enhanceosomes then activate transcription synergistically via interaction with coactivator complexes. The enhanceosome and coactivator complexes are emerging as cellular organizing centers, which integrate signals from a wide range of inputs to generate specific regulatory responses. Knowledge of the mechanisms underlying these important steps in gene control will provide broad insights into biological regulation. [unreadable] [unreadable] This proposal focuses largely on the biochemical mechanisms of activator-coactivator interactions and their role in assembling transcription complexes. We hypothesize that a higher order assembly composed of activators and two coactivators, termed the DA (TFIID and TFIIA) and Med (Mediator) complexes, serves as a key intermediate in transcription initiation. In Aim #1 we test how this so-called DAMed complex is involved in the mechanism of transcriptional synergy and action at a distance by using cell-free mammalian systems and two model activators, GAL4-VP16 and ZEBRA. Aim #2 explores the mechanism and relevance of DAMed complex phosphorylation in initiation. Aim #3 diverges slightly from the main theme of the proposal to examine a prototypical example of specificity by the abundant architectural protein HMGB1 in assembly of ZEBRA-containing enhanceosomes. [unreadable] [unreadable] Our studies are designed to leverage the vast amount of knowledge on the GAL4-VP16 and ZEBRA systems to understand the detailed mechanism of the mediator and enhanceosome in gene control. Both topics are relatively new discoveries, whose understanding will have broad implications on diverse areas of biology. Detailed knowledge of mechanism is necessary to apply concepts derived from studying gene expression to disease-oriented problems. [unreadable] [unreadable]